Canola oil having increased oleic acid and decreased linolenic acid content

ABSTRACT

An endogenous oil extracted from Brassica seeds is disclosed that contains, after crushing and extraction, greater than 86% oleic acid and less than 2.5% α-linolenic acid. The oil also contains less than 7% linoleic acid. The Brassica seeds are produced by plants that contain seed-specific inhibition of microsomal oleate desaturase and microsomal linoleate desaturase gene expression. Such inhibition can be created by cosuppression or antisense technology. Such an oil has a very high oxidative stability in the absence of added antioxidants.

This is a continuation of U.S. application Ser. No. 08/907,608, filedAug. 8, 1997, now U.S. Pat. No. 6,063,947, which is a divisional of U.S.application Ser. No. 08/675,650, filed Jul. 3, 1996, now U.S. Pat. No.5,850,026.

TECHNICAL FIELD

This invention relates to a Brassica canola oil having an elevated oleicacid content and a decreased linolenic acid profile in the seed oil. Theinvention also relates to methods by which such an oil may be produced.

BACKGROUND OF THE INVENTION

Diets high in saturated fats increase low density lipoproteins (LDL)which mediate the deposition of cholesterol on blood vessels. Highplasma levels of serum cholesterol are closely correlated withatherosclerosis and coronary heart disease (Conner et al., CoronaryHeart Disease: Prevention, Complications, and Treatment, pp. 43-64,1985). By producing oilseed Brassica varieties with reduced levels ofindividual and total saturated fats in the seed oil, oil-based foodproducts which contain less saturated fats can be produced. Suchproducts will benefit public health by reducing the incidence ofatherosclerosis and coronary heart disease.

The dietary effects of monounsaturated fats have also been shown to havedramatic effects on health. Oleic acid, the only monounsaturated fat inmost edible vegetable oils, lowers LDL as effectively as linoleic acid,but does not affect high density lipoproteins (HDL) levels (Mattson, F.H., J. Am. Diet. Assoc., 89:387-391, 1989; Mensink et al., New EnglandJ. Med., 321:436-441, 1989). Oleic acid is at least as effective inlowering plasma cholesterol as a diet low in fat and high incarbohydrates (Grundy, S. M., New England J. Med., 314:745-748, 1986;Mensink et al., New England J. Med., 321:436-441, 1989). In fact, a higholeic acid diet is preferable to low fat, high carbohydrate diets fordiabetics (Garg et al., New England J. Med., 319:829-834, 1988). Dietshigh in monounsaturated fats are also correlated with reduced systolicblood pressure (Williams et al., J. Am. Med. Assoc., 257:3251-3256,1987). Epidemiological studies have demonstrated that the“Mediterranean” diet, which is high in fat and monounsaturates, is notassociated with coronary heart disease.

Intensive breeding has produced Brassica plants whose seed oil containsless than 2% erucic acid. The same varieties have also been bred so thatthe defatted meal contains less than 30 μmol glucosinolates/gram.Brassica seeds, or oils extracted from Brassica seeds, that contain lessthan 2% erucic acid (C_(22:1)), and produce a meal with less than 30μmol glucosinolates/gram are referred to as canola seeds or canola oils.Plant lines producing such seeds are also referred to as canola lines orvarieties.

Many breeding studies have been directed to alteration of the fatty acidcomposition in seeds of Brassica varieties. For example, Pleines andFreidt, Fat Sci. Technol., 90(5), 167-171 (1988) describe plant lineswith reduced C_(18:3) levels (2.5-5.8%) combined with high oleic content(73-79%). Roy and Tarr, Z. Pflanzenzuchtg, 95(3), 201-209 (1985) teachestransfer of genes through an interspecific cross from Brassica junceainto Brassica napus resulting in a reconstituted line combining highlinoleic with low linolenic acid content. Roy and Tarr, Plant Breeding,98, 89-96 (1987) discuss prospects for development of B. napus L. havingimproved linolenic and linolenic acid content. Canvin, Can. J. Botany,43, 63-69 (1965) discusses the effect of temperature on the fatty acidcomposition of oils from several seed crops including rapeseed.

Mutations can be induced with extremely high doses of radiation and/orchemical mutagens (Gaul, H. Radiation Botany (1964) 4:155-232). Highdose levels which exceed LD50, and typically reach LD90, led to maximumachievable mutation rates. In mutation breeding of Brassica varieties,high levels of chemical mutagens alone or combined with radiation haveinduced a limited number of fatty acid mutations (Rakow, G. Z.Pflanzenzuchtg (1973) 69:62-82).

Rakow and McGregor, J. Amer. Oil Chem. Soc., 50, 400-403 (October 1973)discuss problems associated with selecting mutants affecting seedlinoleic and linolenic acid levels. The low α-linolenic acid mutationderived from the Rakow mutation breeding program did not have directcommercial application because of low seed yield. The first commercialcultivar using the low α-linolenic acid mutation derived in 1973 wasreleased in 1988 as the variety Stellar (Scarth, R. et al., Can. J.Plant Sci. (1988) 68:509-511). The α-linolenic acid content of Stellarseeds was greater than 3% and the linoleic acid content was about 28%.

Chemical and/or radiation mutagenesis has been used in an attempt todevelop an endogenous canola oil having an oleic acid content of greaterthan 79% and an α-linolenic acid content of less than 5%. Wong, et al.,EP 0 323 753 B1. However, the lowest α-linolenic acid level achieved wasabout 2.7%. PCT publication WO 91/05910 discloses mutagenesis of astarting Brassica napus line in order to increase the oleic acid contentin the seed oil. However, the oleic acid content in canola oil extractedfrom seeds of such mutant lines did not exceed 80%.

The quality of canola oil and its suitability for different end uses isin large measure determined by the relative proportion of the variousfatty acids present in the seed triacylglycerides. As an example, theoxidative stability of canola oil, especially at high temperatures,decreases as the proportion of tri-unsaturated acids increases.Oxidative stability decreases to a lesser extent as the proportion ofdi-unsaturated acids increases. However, it has not been possible toalter the fatty acid composition in Brassica seeds beyond certainlimits. Thus, an endogenous canola oil having altered fatty acidcompositions in seeds is not available for certain specialty uses.Instead, such specialty oils typically are prepared from canola oil byfurther processing, such as hydrogenation and/or fractionation.

SUMMARY OF THE INVENTION

An endogenous oil obtained from Brassica seeds is disclosed. The oil hasan oleic acid content of greater than about 80%, an α-linolenic acidcontent of less than about 2.5% and an erucic acid content of less thanabout 2%, which contents are determined after hydrolysis of the oil.Preferably the oleic acid content is from about 84% to about 88% and theα-linolenic acid content is from about 1% to about 2%. The oil mayfurther have a linoleic acid content of from about 1% to about 10%, alsodetermined after hydrolysis of the oil. The oil can be obtained fromBrassica napus seeds, for example.

Also disclosed herein is a Brassica plant containing at least onerecombinant nucleic construct. The construct(s) comprise a firstseed-specific regulatory sequence fragment operably linked to awild-type microsomal delta-12 fatty acid desaturase coding sequencefragment and a second seed-specific regulatory sequence fragmentoperably linked to a wild-type microsomal delta-15 fatty acid desaturasecoding sequence fragment. Such a plant produces seeds that yield an oilhaving an oleic acid content of about 86% or greater and an erucic acidcontent of less than about 2%, which are determined after hydrolysis ofthe oil. In some embodiments, the plant contains first and secondrecombinant nucleic acid constructs, the first construct comprising thedelta-12 desaturase coding sequence fragment and the second recombinantnucleic acid construct comprising the delta-15 desaturase codingsequence fragment. The delta-12 or delta-15 desaturase coding sequencefragments may comprise either a partial or a full-length Brassicadelta-12 or delta-15 desaturase coding sequence.

Another Brassica plant containing at least one recombinant nucleic acidconstruct is disclosed herein. The construct(s) comprises a firstseed-specific regulatory sequence fragment operably linked to awild-type microsomal delta-12 fatty acid desaturase coding sequencefragment and a second seed-specific regulatory sequence fragmentoperably linked to a wild-type microsomal delta-15 fatty acid desaturasecoding sequence fragment. The plant produces seeds yielding an oilhaving an oleic acid content of 80% or greater, an α-linolenic acidcontent of about 2.5% or less and an erucic acid content of less thanabout 2%, which contents are determined after hydrolysis of the oil.Such a plant may have first and second regulatory sequence fragmentslinked in sense orientation to the delta-12 and delta-15 desaturasecoding sequence fragments, respectively. Alternatively the first andsecond regulatory sequence fragments may be linked in antisenseorientation to the corresponding coding sequence fragments. The delta-12or delta-15 desaturase coding sequence fragments may comprise a partialor a full-length Brassica delta-12 or delta-15 desaturase codingsequence. The plant may produce seeds yielding an oil having an oleicacid content of 80% or greater, an α-linolenic acid content of about2.5% or less and an erucic acid content of less than about 2%, whichcontents are determined after hydrolysis of the oil.

A method of producing an endogenous oil from Brassica seeds is disclosedherein. The method comprises the steps of: creating at least oneBrassica plant having a seed-specific reduction in microsomal delta-12fatty acid desaturase gene expression and a seed-specific reduction inmicrosomal delta-15 fatty acid desaturase gene expression; crushingseeds produced from the plant; and extracting the oil from the seeds.The oil has an oleic acid content of about 86% or greater and an erucicacid content of less than about 2%, determined after hydrolysis of theoil. The seed-specific reduction in delta-12 or delta-15 desaturaseexpression may be created by cosuppression or antisense.

Another method of producing an endogenous oil from Brassica seeds isdisclosed herein. The method comprises the steps of: creating at leastone Brassica plant having a seed-specific reduction in microsomaldelta-12 fatty acid desaturase gene expression and a seed-specificreduction in microsomal delta-15 fatty acid desaturase gene expression;crushing seeds produced from the plant; and extracting the oil from theseeds. The oil has an oleic acid content of about 80% or greater, anα-linolenic acid content of 2.5% or less and an erucic acid content ofless than about 2%, determined after hydrolysis of the oil. Theseed-specific reduction in delta-12 or delta-15 desaturase expressionmay be created by cosuppression or by antisense.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The term “fatty acid desaturase” refers to an enzyme which catalyzes thebreakage of a carbon-hydrogen bond and the introduction of acarbon-carbon double bond into a fatty acid molecule. The fatty acid maybe free or esterified to another molecule including, but not limited to,acyl-carrier protein, coenzyme A, sterols and the glycerol moiety ofglycerolipids. The term “glycerolipid desaturases” refers to a subset ofthe fatty acid desaturases that act on fatty acyl moieties esterified toa glycerol backbone. “Delta-12 desaturase” refers to a fatty aciddesaturase that catalyzes the formation of a double bond between carbonpositions 6 and 7 (numbered from the methyl end), (i.e., those thatcorrespond to carbon positions 12 and 13 (numbered from the carbonylcarbon) of an 18 carbon-long fatty acyl chain. “Delta-15 desaturase”refers to a fatty acid desaturase that catalyzes the formation of adouble bond between carbon positions 3 and 4 (numbered from the methylend), (i.e., those that correspond to carbon positions 15 and 16(numbered from the carbonyl carbon) of an 18 carbon-long fatty acylchain. “Microsomal desaturase” refers to the cytoplasmic location of theenzyme, while “chloroplast desaturase” and “plastid desaturase” refer tothe plastid location of the enzyme. It should be noted that these fattyacid desaturases have never been isolated and characterized as proteins.Accordingly, the terms such as “delta-12 desaturase” and “delta-15desaturase” are used as a convenience to describe the proteins encodedby nucleic acid fragments that have been isolated based on thephenotypic effects caused by their disruption. They do not imply anycatalytic mechanism. For example, delta-12 desaturase refers to theenzyme that catalyzes the formation of a double bond between carbons 12and 13 of an 18 carbon fatty acid irrespective of whether it “counts”the carbons from the methyl, carboxyl end, or the first double bond.

Microsomal delta-12 fatty acid desaturase (also known as omega-6 fattyacid desaturase, cytoplasmic oleic desaturase or oleate desaturase) isinvolved in the enzymatic conversion of oleic acid to linoleic acid. Amicrosomal delta-12 desaturase has been cloned and characterized usingT-DNA tagging. Okuley, et al., Plant Cell 6:147-158 (1994). Thenucleotide sequences of higher plant genes encoding microsomal delta-12fatty acid desaturase are described in Lightner et al., WO94/11516.

Microsomal delta-15 fatty acid desaturase (also known as omega-3 fattyacid desaturase, cytoplasmic linoleic acid desaturase or linoleatedesaturase) is involved in the enzymatic conversion of linoleic acid toα-linolenic acid. Sequences of higher plant genes encoding microsomaland plastid delta-15 fatty acid desaturases are disclosed in Yadav, N.,et al., Plant Physiol., 103:467-476 (1993), WO 93/11245 and Arondel, V.et al., Science, 258:1353-1355 (1992).

Brassica species have more than one gene for endogenous microsomaldelta-12 desaturase and more than one gene for endogenous microsomaldelta-15 desaturase. The genes for microsomal delta-12 desaturase aredesignated Fad2 while the genes for microsomal delta-15 desaturase aredesignated Fad3. In amphidiploids, each gene is derived from one of theancestral genomes making up the species under consideration. Thefull-length coding sequences for the wild-type Fad2 genes from Brassicanapus (termed the D form and the F form) are shown in SEQ ID NO:1 andSEQ ID NO:5, respectively. The full-length coding sequence for awild-type Fad3 gene is disclosed in WO 93/11245.

The inventors have discovered canola oils that have novel fatty acidcompositions, e.g., very high oleic acid levels and very low α-linolenicacid levels. Such oils may be obtained by crushing seeds of transgenicBrassica plants exhibiting a seed-specific reduction in delta-12desaturase and delta-15 desaturase activity; oil of the invention isextracted therefrom. Expression of Fad2 and Fad3 in seeds is reducedsuch that the resulting seed oil possesses very high levels of oleicacid and very low levels of α-linolenic acid. The fatty acid compositionof the endogenous seed oil, as determined after hydrolysis of fatty acidesters reflects the novel fatty acid composition of such seeds.

The fatty acid composition of oils disclosed herein is determined bytechniques known to the skilled artisan, e.g., hydrolysis of esterifiedfatty acids (triacylglycerides and the like) in a bulk seed samplefollowed by gas-liquid chromatography (GLC) analysis of fatty acidmethyl esters.

In one embodiment, an oil of the invention has an oleic acid content ofabout 80% or greater, as well as a surprisingly low α-linolenic acidcontent of about 2.5% or less. The oleic acid content is preferably fromabout 84% to about 89%, more preferably from about 86% to about 89%. Theα-linolenic acid preferably is from about 1% to less than about 2.5%,more preferably from about 1% to about 2%.

The linoleic acid content of an oil of this embodiment typically is lessthan about 10%, preferably less than about 7%, more preferably fromabout 2% to about 6%.

Canola seed is crushed by techniques known in the art. The seedtypically is tempered by spraying the seed with water to raise themoisture to, for example, 8.5%. The tempered seed is flaked using smoothroller with, for example, a gap setting of 0.23 to 0.27 mm. Heat may beapplied to the flakes to deactivate enzymes, facilitate further cellrupturing, coalesce the oil droplets and agglomerate protein particlesin order to ease the extraction process.

Typically, oil is removed from the heated canola flakes by a screw pressto press out a major fraction of the oil from the flakes. The resultingpress cake contains some residual oil.

Crude oil produced from the pressing operation typically is passedthrough a settling tank with a slotted wire drainage top to remove thesolids expressed out with the oil in the screw pressing operation. Theclarified oil can be passed through a plate and frame filter to removethe remaining fine solid particles.

Canola press cake produced from the screw pressing operation can beextracted with commercial n-Hexane. The canola oil recovered from theextraction process is combined with the clarified oil from the screwpressing operation, resulting in a blended crude oil.

Free fatty acids and gums typically are removed from the crude oil byheating in a batch refining tank to which food grade phosphoric acid hasbeen added. The acid serves to convert the non-hydratable phosphatidesto a hydratable form, and to chelate minor metals that are present inthe crude oil. The phosphatides and the metal salts are removed from theoil along with the soapstock. The oil-acid mixture is treated withsodium hydroxide solution to neutralize the free fatty acids and thephosphoric acid in the acid-oil mixture. The neutralized free fattyacids, phosphatides, etc. (soapstock) are drained off from theneutralized oil. A water wash may be done to further reduce the soapcontent of the oil. The oil may be bleached and deodorized before use,if desired, by techniques known in the art.

A transgenic plant disclosed herein contains at least one recombinantnucleic acid construct. The construct or constructs comprise an oleatedesaturase coding sequence fragment and a linoleate desaturase codingsequence fragment, both of which are expressed preferentially indeveloping seeds. Seed-specific expression of the recombinantdesaturases results in a seed-specific reduction in native desaturasegene expression. The seed-specific defect in delta-12 and delta-15desaturase gene expression alters the fatty acid composition in matureseeds produced on the plant, so that the oil obtained from such seedshas the novel fatty acid compositions disclosed herein.

Typically, the oleate and linoleate desaturase sequence fragments arepresent on separate constructs and are introduced into thenon-transgenic parent on separate plasmids. The desaturase fragments maybe isolated or derived from, e.g., Brassica spp., soybean (Glycine max),sunflower and Arabidopsis. Preferred host or recipient organisms forintroduction of a nucleic acid construct are oil-producing species, suchas Brassica napus, B. rapa and B. juncea.

A transgenic plant disclosed herein preferably is homozygous for thetransgene containing construct. Such a plant may be used as a parent todevelop plant lines or may itself be a member of a plant line, i.e., beone of a group of plants that display little or no genetic variationbetween individuals for the novel oil composition trait. Such lines maybe created by several generations of self-pollination and selection, orvegetative propagation from a single parent using tissue or cell culturetechniques. Other means of breeding plant lines from a parent plant areknown in the art.

Progeny of a transgenic plant are included within the scope of theinvention, provided that such progeny exhibit the novel seed oilcharacteristics disclosed herein. Progeny of an instant plant include,for example, seeds formed on F₁, F₂, F₃, and subsequent generationplants, or seeds formed on BC₁, BC₂, BC₃ and subsequent generationplants.

A seed-specific reduction in Fad2 and Fad3 gene expression may beachieved by techniques including, but not limited to, antisense andcosuppression. These phenomena significantly reduce expression of thegene product by the native genes (wild-type or mutated). The reductionin gene expression can be inferred from the decreased level of reactionproduct and the increased level of substrate in seeds (e.g., decreased18:2 and increased 18:1), compared to the corresponding levels in planttissues expressing the native genes.

The preparation of antisense and cosuppression constructs for inhibitionof fatty acid desaturases may utilize fragments containing thetranscribed sequence for the Fad2 and Fad3 fatty acid desaturase genesin canola. These genes have been cloned and sequenced as discussedhereinabove.

Antisense RNA has been used to inhibit plant target genes in atissue-specific manner. van der Krol et al., Biotechniques 6:958-976(1988). Antisense inhibition has been shown using the entire cDNAsequence as well as a partial cDNA sequence. Sheehy et al., Proc. Natl.Acad. Sci. USA 85:8805-8809 (1988); Cannon et al., Plant Mol. Biol.15:39-47 (1990). There is also evidence that 3′ non-coding sequencefragment and 5′ coding sequence fragments, containing as few as 41base-pairs of a 1.87 kb cDNA, can play important roles in antisenseinhibition. (Ch'ng et al., Proc. Natl. Acad. Sci. USA 86:10006-10010(1989); Cannon et al., supra.

The phenomenon of cosuppression has also been used to inhibit planttarget genes in a tissue-specific manner. Cosuppression of an endogenousgene using a full-length cDNA sequence as well as a partial cDNAsequence (730 bp of a 1770 bp cDNA) are known. Napoli et al., The PlantCell 2:279-289 (1990); van der Krol et al., The Plant Cell 2:291-299(1990); Smith et al., Mol. Gen. Genetics 224:477-481 (1990).

Nucleic acid fragments comprising a partial or a full-length delta-12 ordelta-15 fatty acid desaturase coding sequence are operably linked to atleast one suitable regulatory sequence in antisense orientation (forantisense constructs) or in sense orientation (for cosuppressionconstructs). Molecular biology techniques for preparing such chimericgenes are known in the art. The chimeric gene is introduced into aBrassica plant and transgenic progeny displaying a fatty acidcomposition disclosed herein due to antisense or cosuppression areidentified. Transgenic plants that produce a seed oil having a fattyacid composition disclosed herein are selected for use in the invention.Experimental procedures to develop and identify cosuppressed plantsinvolve breeding techniques and fatty acid analytical techniques knownin the art.

One may use a partial cDNA sequence for cosuppression as well as forantisense inhibition. For example, cosuppression of delta-12 desaturaseand delta-15 desaturase in Brassica napus may be achieved by expressing,in the sense orientation, the entire or partial seed delta-12 desaturasecDNA found in pCF2-165D. See WO 04/11516.

Seed-specific expression of native Fad2 and Fad3 genes can also beinhibited by non-coding regions of an introduced copy of the gene. See,e.g., Brusslan, J. A. et al. (1993) Plant Cell 5:667-677; Matzke, M. A.et al., Plant Molecular Biology 16:821-830). One skilled in the art canreadily isolate genomic DNA containing sequences that flank desaturasecoding sequences and use the non-coding regions for antisense orcosuppression inhibition.

Regulatory sequences typically do not themselves code for a geneproduct. Instead, regulatory sequences affect the expression level ofthe mutant coding sequence. Examples of regulatory sequences are knownin the art and include, without limitation, promoters of genes expressedduring embryogenesis, e.g., a napin promoter, a phaseolin promoter, aoleosin promoter and a cruciferin promoter. Native regulatory sequences,including the native promoters, of delta-12 and delta-15 fatty aciddesaturase genes can be readily isolated by those skilled in the art andused in constructs of the invention. Other examples of suitableregulatory sequences include enhancers or enhancer-like elements,introns and 3′ non-coding regions such as poly A sequences. Furtherexamples of suitable regulatory sequences for the proper expression ofmutant or wild-type delta-12 or mutant delta-15 coding sequences areknown in the art.

In preferred embodiments, regulatory sequences are seed-specific, i.e.,the chimeric desaturase gene product is preferentially expressed indeveloping seeds and expressed at low levels or not at all in theremaining tissues of the plant. Seed-specific regulatory sequencespreferably stimulate or induce expression of the recombinant desaturasecoding sequence fragment at a time that coincides with or slightlyprecedes expression of the native desaturase gene. Murphy et al., J.Plant Physiol. 135:63-69 (1989).

Transgenic plants for use in the invention are created by transformingplant cells of Brassica species. Such techniques include, withoutlimitation, Agrobacterium-mediated transformation, electroporation andparticle gun transformation. Illustrative examples of transformationtechniques are described in U.S. Pat. No. 5,204,253, (particle gun) andU.S. Pat. No. 5,188,958 (Agrobacterium), incorporated herein byreference. Transformation methods utilizing the Ti and Ri plasmids ofAgrobacterium spp. typically use binary type vectors. Walkerpeach, C. etal., in Plant Molecular Biology Manual, S. Gelvin and R. Schilperoort,eds., Kluwer Dordrecht, C1:1-19 (1994). If cell or tissue cultures areused as the recipient tissue for transformation, plants can beregenerated from transformed cultures by techniques known to thoseskilled in the art.

One or more recombinant nucleic acid constructs, suitable for antisenseor cosuppression of native Fad2 and Fad3 genes are introduced, and atleast one transgenic Brassica plant is obtained. Seeds produced by thetransgenic plant(s) are grown and either selfed or outcrossed to obtainplants homozygous for the recombinant construct. Seeds are analyzed asdiscussed above in order to identify those homozygotes having nativefatty acid desaturase activities inhibited by the mechanisms discussedabove. Homozygotes may be entered into a breeding program, e.g., toincrease seed, to introgress the novel oil composition trait into otherlines or species, or for further selection of other desirable traits(disease resistance, yield and the like).

Fatty acid composition is followed during the breeding program byanalysis of a bulked seed sample or of a single half-seed. Half-seedanalysis useful because the viability of the embryo is maintained andthus those seeds having a desired fatty acid profile may be advanced tothe next generation. However, half-seed analysis is also known to be aninaccurate representation of the genotype of the seed being analyzed.Bulk seed analysis typically yields a more accurate representation ofthe fatty acid profile in seeds of a given genotype.

Procedures for analysis of fatty acid composition are known in the art.These procedures can be used to identify individuals to be retained in abreeding program; the procedures can also be used to determine theproduct specifications of commercial or pilot plant oils.

The relative content of each fatty acid in canola seeds can bedetermined either by direct transesterification of individual seeds inmethanolic H₂SO₄ (2.5%) or by hexane extraction of bulk seed samplesfollowed by trans-esterification of an aliquot in 1% sodium methoxide inmethanol. Fatty acid methyl esters can be extracted from the methanolicsolutions into hexane after the addition of an equal volume of water.

For example, a seed sample from each transformant in a breeding programis crushed with a mortar and pestle and extracted 4 times with 8 mLhexane at about 50° C. The extracts from each sample are reduced involume and two aliquots are taken for esterification. Separation of thefatty acid methyl esters can be carried out by gas-liquid chromatographyusing an Omegawax 320 column (Supelco Inc., 0.32 mm ID×30M) runisothermally at 220° and cycled to 260° between each injection.

Alternatively, seed samples from a breeding program are ground andextracted in methanol/KOH, extracted with iso-octane, and fatty acidsseparated by gas chromatography.

A method to produce an oil of the invention comprises the creation of atleast one Brassica plant having a seed-specific reduction in Fad2 andFad3 gene expression, as discussed above. Seeds produced by such aplant, or its progeny, are crushed and the oil is extracted from thecrushed seeds. Such lines produce seeds yielding an oil of theinvention, e.g., an oil having from about 80% to about 88% oleic acid,from about 1% to about 2% α-linolenic acid and less than about 2% erucicacid.

Alternatively, such a plant can be created by crossing two parentplants, one exhibiting a reduction in Fad2 gene expression and the otherexhibiting a reduction in Fad3 gene expression. Progeny of the cross areoutcrossed or selfed in order to obtain progeny seeds homozygous forboth traits.

Transgenic plants having a substantial reduction in Fad2 and Fad3 geneexpression in seeds have novel fatty acid profiles in oil extracted fromsuch seeds, compared to known canola plants, e.g., the reduction in bothdesaturase activities results in a novel combination of high oleic andlower α-linolenic acid in seed oils. By combining seed-specificinhibition of microsomal delta-12 desaturase with seed-specificinhibition of microsomal delta-15 desaturase, one obtains very lowlevels of seed α-linolenic acid, without adversely affecting agronomicproperties.

It is noteworthy that Fad2 and Fad3 cosuppression constructs provide anovel means for producing canola oil having 86% oleic acid or greater. Amethod of producing a canola oil having greater than 86% oleic acidcomprises the creation of a transgenic Brassica plant containing atleast one recombinant nucleic acid construct, which construct(s)comprises an oleate desaturase coding sequence expressed preferentiallyin developing seeds and a linoleate desaturase coding sequence expressedpreferentially in developing seeds. A proportion of the plants that arehomozygous for the transgenes have seed-specific cosuppression of thenative linoleate desaturase. Seeds produced by such transgeniccosuppressed plants are crushed and the oil is extracted therefrom. Theoil has about 86% or greater oleic acid and less than about 2% erucicacid. The oleic acid content can be as high as 89%.

Transgenic plants exhibiting cosuppression of Fad2 and Fad3 produceseeds having a very high oleic acid content. This result was unexpectedbecause it was not known if one could obtain plants in which inhibitionof Fad2 and Fad3 via cosuppression was sufficient to achieve an oleicacid level of 86% or greater in seeds. Indeed, it was not known if twocosuppressed genes in fatty acid metabolism could be introduced incanola without the first cosuppression gene interfering with the secondcosuppression gene, or without adversely affecting other agronomictraits.

Marker-assisted breeding techniques may be used to identify and follow adesired fatty acid composition during the breeding process. Such markersmay include RFLP, RAPD, or PCR markers, for example. Marker-assistedbreeding techniques may be used in addition to, or as an alternative to,other sorts of identification techniques. An example of marker-assistedbreeding is the use of PCR primers that specifically amplify thejunction between a promoter fragment and the coding sequence of a Fad2gene.

While the invention is susceptible to various modifications andalternative forms, certain specific embodiments thereof are described inthe general methods and examples set forth below. For example theinvention may be applied to all Brassica species, including B. rapa, B.juncea, and B. hirta, to produce substantially similar results. Itshould be understood, however, that these examples are not intended tolimit the invention to the particular forms disclosed. Instead, thedisclosure is to cover all modifications, equivalents and alternativesfalling within the scope of the invention.

EXAMPLE 1 CONSTRUCTS FOR COSUPPRESSION OF DELTA-12 FATTY ACID DESATURASEAND DELTA-15 FATTY ACID DESATURASE

The wild-type Brassica cDNA coding sequence for the delta-12 desaturaseD form was cloned as described in WO 94/11516, which is incorporatedherein by reference. Briefly, rapeseed cDNAs encoding cytoplasmic oleate(18:1) desaturase were obtained by screening a cDNA library made fromdeveloping rapeseed using a heterologous probe derived from anArabidopsis cDNA fragment encoding the same enzyme. (Okuley et al 1994).The full-length coding sequence of Fad2 is found as SEQ ID NO:1.Rapeseed cDNAs encoding the cytoplasmic linoleate (18:2) desaturase(Fad3) were obtained as described in WO 93/11245, incorporated herein byreference. See also (Yadav et. al 1993). Seed specific expression ofthese cDNAs in transgenic rapeseed was driven by one of four differentseed storage protein promoters, napin, oleosin and cruciferin promotersfrom B. napus and a phaseolin promoter from Phaseolus vulgaris.

Detailed procedures for manipulation of DNA fragments by restrictionendonuclease digestion, size separation by agarose gel electrophoresis,isolation of DNA fragments from agarose gels, ligation of DNA fragments,modification of cut ends of DNA fragments and transformation of E. colicells with plasmids have been described. Sambrook et al., (MolecularCloning, A Laboratory Manual, 2nd ed (1989) Cold Spring HarborLaboratory Press); Ausubel et al., Current Protocols in MolecularBiology (1989) John Wiley & Sons). Plant molecular biology proceduresare described in Plant Molecular Biology Manual, Gelvin S. andSchilperoort, R. eds. Kluwer, Dordrecht (1994).

The plasmid pZS212 was used to construct binary vectors for theseexperiments. pZS212 contains a chimeric CaMV35S/NPT gene for use inselecting kanamycin resistant transformed plant cells, the left andright border of an Agrobacterium Ti plasmid T-DNA, the E. coli lacZ α-)complementing segment with unique restriction endonuclease sites forEcoRI, KpnI, BamHI and SalI, the bacterial replication origin from thePseudomonas plasmid pVS1 and a bacterial Tn5 NPT gene for selection oftransformed Agrobacterium. See WO 94/11516, p. 100.

The first construct was prepared by inserting a full-length mutantBrassica Fad2 D gene coding sequence fragment in sense orientationbetween the phaseolin promoter and phaseolin 3′ poly A region of plasmidpCW108. The full-length coding sequence of the mutant gene is found inSEQ ID NO:3.

The pCW108 vector contains the bean phaseolin promoter and 3′untranslated region and was derived from the commercially availablepUC18 plasmid (Gibco-BRL) via plasmids AS3 and pCW104. Plasmid AS3contains 495 base pairs of the Phaseolus vulgaris phaseolin promoterstarting with 5′-TGGTCTTTTGGT-3′ followed by the entire 1175 base pairsof the 3′ untranslated region of the same gene. Sequence descriptions ofthe 7S seed storage protein promoter are found in Doyle et al., J. Biol.Chem. 261:9228-9238 (1986) and Slightom et al., Proc. Natl. Acad. Sci.USA, 80:1897-1901 (1983). Further sequence description may be found inWO 91/13993. The fragment was cloned into the HindIII site of pUC18. Theadditional cloning sites of the pUC18 multiple cloning region (Eco RI,SphI, PstI and SalI) were removed by digesting with Eco RI and SalI,filling in the ends with Klenow and religating to yield the plasmidpCW104. A new multiple cloning site was created between the 495 bp ofthe 5′ phaseolin and the 1175 bp of the 3′ phaseolin by inserting adimer of complementary synthetic oligonucleotides to create the plasmidpCW108. See WO 94/11516. This plasmid contains unique NcoI, SmaI, KpnIand XbaI sites directly behind the phaseolin promoter.

The phaseolin promoter:mutantFad2:phaseolin poly A construct in pCW108was excised and cloned between the SalI/EcoRI sites of pZS212. Theresulting plasmid was designated pIMC201.

A second plasmid was constructed by inserting the full-length wild typeBrassica Fad2 D gene coding sequence into the NotI site of plasmidpIMC401, which contains a 2.2 kb napin expression cassette. See, e.g.,WO94/11516, page 102. The 5′-napin:Fad2:napin poly A-3′ construct wasinserted into the SalI site of pZS212 and the resulting 17.2 Kb plasmidwas termed pIMC127. Napin promoter sequences are also disclosed in U.S.Pat. No. 5,420,034.

A third plasmid, pIMC135, was constructed in a manner similar to thatdescribed above for pIMC127. Plasmid pIMC135 contains a 5′ cruciferinpromoter fragment operably linked in sense orientation to thefull-length wild-type Brassica Fad2 D gene coding sequence, followed bya cruciferin 3′ poly A fragment. The 5′-cruciferin:Fad2 D:cruciferinpolyA cassette was inserted into pZS212; the resulting plasmid wastermed pIMC135. Suitable cruciferin regulatory sequences are disclosedin Rodin, J. et al., J. Biol. Chem. 265:2720 (1990); Ryan, A. et al.,Nucl. Acids Res. 17:3584 (1989) and Simon, A. et al., Plant Mol. Biol.5:191 (1985). Suitable sequences are also disclosed in the Genbankcomputer database, e.g., Accession No. M93103.

A fourth plasmid, pIMC133 was constructed in a manner similar to thatdescribed above. Plasmid pIMC133 contains a 5′ oleosin promoter fragmentoperably linked in sense orientation to the full-length Brassica Fad2 Dgene coding sequence, followed by a 3′ oleosin poly A fragment. See,e.g., WO 93/20216, incorporated herein by reference.

A napin-Fad3 construct was made by first isolating a delta-15 desaturasecoding sequence fragment from pBNSF3-f2. The fragment contained thefull-length coding sequence of the desaturase, disclosed as SEQ ID NO: 6in WO 93/11245, incorporated herein by reference. The 1.2 kb fragmentwas fitted with linkers and ligated into pIMC401. The5′napin:Fad3:3′napin cassette was inserted into the SalI site of pZS212;the resulting plasmid was designated pIMC110.

EXAMPLE 2 CREATION OF TRANSGENIC COSUPPRESSED PLANTS

The plasmids pIMC201, pIMC127, pIMC135, pIMC133 and pIMC110 wereintroduced into Agrobacterium strain LBA4404/pAL4404 by a freeze-thawmethod. The plasmids were introduced into Brassica napus cultivar Westarby the method of Agrobacterium-mediated transformation as described inWO94/11516, incorporated herein by reference. Transgenic progeny plantscontaining pIMC201 were designated as the WS201 series. Plantstransformed with pIMC127 were designated as the WS687 series. Plantstransformed with pIMC135 were designated as the WS691 series. Plantstransformed with pIMC133 were designated as the WS692 series. Plantstransformed with pIMC110 were designated as the WS663 series.

Unless indicated otherwise, fatty acid percentages described herein arepercent by weight of the oil in the indicated seeds as determined afterextraction and hydrolysis.

From about 50 to 350 transformed plants (T1 generation) were producedfor each cDNA and promoter combination. T1 plants were selfed to obtainT2 seed. T2 samples in which cosuppression events occurred wereidentified from the fatty acid profile and from the presence of thetransgene by molecular analysis. The transformed plants were screenedfor phenotype by analysis of the relative fatty acid contents of bulkseed from the first transformed generation by GC separation of fattyacid methyl esters.

T2 seed was sown in 4-inch pots containing Pro-Mix soil. The plants,along with Westar controls, were grown at 25±3° C./18±3° C., 14/10 hrday/night conditions in the greenhouse. At flowering, the terminalraceme was self-pollinated by bagging. At maturity, seed wasindividually harvested from each plant, labelled, and stored to ensurethat the source of the seed was known.

Fatty acid profiles were determined as described in WO 91/05910. Forchemical analysis, 10-seed bulk samples were hand ground with a glassrod in a 15-mL polypropylene tube and extracted in 1.2 mL 0.25 N KOH in1:1 ether/methanol. The sample was vortexed for 30 sec. and heated for60 sec. in a 60° C. water bath. Four mL of saturated NaCl and 2.4 mL ofiso-octane were added, and the mixture was vortexed again. After phaseseparation, 600 μL of the upper organic phase were pipetted intoindividual vials and stored under nitrogen at −5° C. One μL samples wereinjected into a Supelco SP-2330 fused silica capillary column (0.25 mmID, 30 M length, 0.20 μm df).

The gas chromatograph was set at 180° C. for 5.5 minutes, thenprogrammed for a 2° C./minute increase to 212° C., and held at thistemperature for 1.5 minutes. Total run time was 23 minutes.Chromatography settings were: Column head pressure—15 psi, Column flow(He)—0.7 mL/min., Auxiliary and Column flow—33 mL/min., Hydrogen flow—33mL/min., Air flow—400 mL/min., Injector temperature—250° C., Detectortemperature—300° C., Split vent—1/15.

Table 1 shows the content of the seven major fatty acids in mature seedsfrom transgenic cosuppressed plants homozygous for the napin:Fad3construct or the napin:Fad2 construct (T4 or later generation). Overexpression phenotypes and cosuppression phenotypes were observed forboth chimeric genes (oleate desaturase and linoleate desaturase); datafor plants exhibiting the cosuppression phenotype are shown in theTable.

As shown in Table 1, the homozygous Fad2-cosuppressed seed had aα-linolenic acid content of about 2.9%, which was less than half that ofthe Westar control; the oleic acid content increased to about 84.1%. Thehomozygous Fad3-cosuppressed seed had an α-linolenic acid of about 1.2%;the oleic acid and linoleic acid contents in Fad3-cosuppressed plantsincreased slightly compared to Westar. The results demonstrate thatinhibiting gene expression of either enzyme by cosuppression resulted ina change in fatty acid composition of the seed oil.

TABLE 1 Fatty Acid Profiles in Oil From Cosuppression Canola SeedTRANSGENE FATTY ACID (% OF TOTAL FATTY ACIDS) CONSTRUCTION 16:0 18:018:1 18:2 18:3 20:0 20:1 22:0 24:0 non-transformed 3.9 1.8 67.0 19.0 7.50.6 0.8 0.6 0.1 Westar napin:Fad2 4.3 1.4 84.1  5.2 2.9 0.6 0.9 0.5 0.2(co-suppression) napin:Fad3 3.8 1.5 68.5 22.1 1.2 0.6 1.1 0.4 0.1(co-suppression)

TABLE 2 Fatty Acid Profiles in Oil From Cosuppression Canola SeedsConstruct (promoter/coding Fatty Acid Composition Line # sequence) 16:018:0 18:1 18:2 18:3 663-40 napin/Fad3 3.9 1.4 71.2 20.1 1.2 687-193napin/Fad2 4.0 1.5 82.8  5.9 3.7 691-215 cruciferin/Fad2 3.3 1.3 86.5 3.0 3.7 692-090-3 oleosin/Fad2 3.4 1.3 86.5  2.6 3.9 692-105-11oleosin/Fad2 3.4 1.3 86.2  2.7 4.2 201-389 A23 phaseolin/MFad2 4.2 2.784.6  4.7 3.7

TABLE 3 Range of Fatty Acid Profiles for Fad2 and Fad3 CosuppressionLines Tested in the Field Fatty Acid Composition Line No. Vector Min/MaxC16:0 C18:0 C18:1 C18:2 C18:3 663-40 pIMC110 Min 3.5 2.3 73.5 16.3 0.8Max 4.7 2.2 64.0 24.2 1.5 687-193 pIMC127 Min 3.4 3.1 83.3  3.8 2.3 Max3.4 2.1 85.5  3.2 2.5 692-105 pIMC133 Min 3.7 2.7 84.6  2.8 2.4 Max 3.32.3 86.3  2.1 2.7 691-215 pIMC135 Min 3.2 2.4 84.6  3.0 2.5 Max 3.0 2.086.3  2.6 2.5

Table 2 shows the fatty acid profile in T4 or later homozygous seedsproduced by six individual plants having various promoter-desaturasegene combinations. The seeds were obtained from greenhouse-grown plants.The results indicate that the oleic acid content ranged from about 82.8%to about 86.5% among the lines carrying the Fad2 constructs. Thephaseolin:mutated Fad2 construct was as successful as the wild-type Fad2constructs in achieving seed-specific Fad2 cosuppression.

The napin:Fad3 cosuppressed plant line had an unusually low α-linolenicacid content of 1.2%. However, the oleic acid content was only 71.2% andthe linoleic acid content was similar to that of the non-transformedcontrol Westar in Table 1.

Homozygous seeds from four of the lines in Table 2 were planted in afield nursery in Colorado and self-pollinated. Seed samples from severalplants of each line were collected and separately analyzed for fattyacid composition. The results for the 663-40 plant having the minimumand the 663-40 plant having the maximum linolenic acid content observedin the field are shown in Table 3. The results for the 687-193, 692-105and 691-215 plants having the minimum and maximum oleic acid content inthe field are also shown in Table 3.

The results in Table 3 demonstrate that the fatty acid profile infield-grown seeds of cosuppressed transgenic plants was similar to thatin the greenhouse-grown seeds (Table 2), indicating that thecosuppression trait confers a stable fatty acid composition on the oil.The results also indicate that an oil having the combination of an oleicacid content of 86% or greater and an α-linolenic acid content of 2.5%or less could not be obtained from plants cosuppressed for either Fad2or Fad3 alone.

EXAMPLE 3 OIL CONTENT IN SEEDS OF PLANTS EXHIBITING Fad2 and Fad3COSUPPRESSION

Crosses were made between the napin:Fad3 cosuppressed line 663-40 andthree Fad2 cosuppressed lines, 691-215, 692-090-3 and 692-105-11. F1plants were selfed for 2 generations in the greenhouse to obtain F3generation seed that was homozygous for both recombinant constructs.

TABLE 4 Fatty Acid Profile in F3 Seeds of Lines Exhibiting Fad2 and Fad3Cosuppression Line # Construct 16:0 18:0 18:1 18:2 18:3 663-40napin/Fad3 3.9 1.4 71.2 20.1 1.2 691-215 cruciferin/Fad2 3.9 1.3 86.5 3.0 3.7 663-40X691-215 napin/Fad3 & 3.2 1.4 86.2  5.2 1.5cruciferin/Fad2 663-40 napin/Fad3 3.9 1.4 71.2 20.1 1.2 692-090-3oleosin/Fad2 3.4 1.3 86.5  2.6 3.9 663-40X692-090-3 napin/Fad3 & 3.4 1.585.5  5.0 1.7 oleosin/Fad2 663-40 napin/Fad3 3.9 1.4 71.2 20.1 1.2692-105-11 oleosin/Fad2 3.4 1.3 86.2  2.7 4.2 663-40X692-105-11napin/Fad3 & 3.4 1.4 86.8  4.6 1.4 oleosin/Fad2

The seed fatty acid profiles of the parent lines and a representative F3cosuppressed line are shown in Table 4. Plants expressing bothcosuppression constructs exhibited an oleic acid level of about 86% orgreater. Moreover, this high level of oleic acid was present incombination with an unusually low level of α-linolenic acid, less than2.0%. However, the linoleic acid content in the F3 seeds increased fromabout 2.6-3.0% to about 4.6-5.2%.

These results demonstrate that a canola oil can be extracted fromrapeseeds that contains greater than 80% oleic acid and less than 2.5%α-linolenic acid. Results similar to those obtained using cosuppressionconstructs are achieved when antisense constructs are used.

The canola oil extracted from Fad2 and Fad3 cosuppressed F3 seed, orprogeny thereof, is found to have superior oxidative stability comparedto the oil extracted from Westar seed. The improved oxidative stabilityof such an oil is measured after refining, bleaching and deodorizing,using the Accelerated oxygen Method (AOM), American Oil Chemists'Society Official Method Cd 12-57 for fat stability, Active Oxygen Method(revised 1989). The improved oxidative stability is also demonstratedwhen using the Oxidative Stability Index method. The improved oxidativestability is measured in the absence of added antioxidants.

EXAMPLE 4 OIL CONTENT IN SEEDS OF PLANTS HAVING Fad3 COSUPPRESSION ANDCHEMICALLY-INDUCED Fad2 MUTATIONS

Q4275 is a doubly mutagenized B. napus line having defects in the Fad2gene. Q4275 was derived by chemical mutagenesis of B. napus line IMC129,which carries a mutation in the Fad2 D gene; the coding sequence of themutated gene is shown in SEQ ID NO:3. Line IMC129 was itself derived bychemical mutagenesis of the cultivar Westar, as disclosed in WO91/05910. Genetic segregation analysis of crosses between Q4275 andother fatty acid mutant lines indicated that Q4275 carried a mutation inthe B. napus Fad2 F gene in addition to the IMC129 Fad2 D gene mutation.Q4275 thus carries chemically induced mutations in both Fad2 genes.

A cross was made between Q4275 and the napin:Fad3 cosuppressed line663-40. F1 plants were selfed in the greenhouse and F2 plants that werehomozygous for the recombinant construct and the Fad2 D and Fad2 Fmutated genes were identified by fatty acid profile analysis of the F3generation seed. After selfing to homozygosity, the fatty acid profilesin seeds of a representative homozygous plant was analyzed and comparedto the profile of the parent plants, as shown in Table 5.

The results show that an oil having greater than 87% oleic acid and lessthan 1.5% α-linolenic acid can be obtained from a transgenic Brassicaplant containing a seed-specific reduction in Fad3 gene expression aswell as chemically-induced mutations in Fad2 genes.

TABLE 5 Fatty Acid Profile of Fad3 Cosuppression, Fad2 Mutated Seeds16:0 18:0 18:1 18:2 18:3 663-40 3.9 1.4 71.2 20.1 1.2 Q4275 3.3 1.5 86.72.2 3.1 Q4275 X 663-40 3.2 1.6 87.6 4.2 1.3

TABLE 6 Range of Fatty Acid Profiles for Fad3 Cosuppression, Fad2Mutated Lines Tested in the Field 16:0 18:0 18:1 18:2 18:3 663-40 Min3.5 2.3 73.5 16.3 0.8 Max 4.7 2.2 64.0 24.2 1.5 Q4275 Min 3.2 3.3 85.01.8 2.0 Max 3.0 2.3 86.6 1.7 2.6 Q4275 X 663-40 Min 3.2 2.0 85.1 5.3 0.9Max 3.2 2.9 84.0 6.0 1.5

Additional seed from the homozygous plant described above was planted inthe field and self-pollinated. Mature seeds from several progeny plantswere separately analyzed for their fatty acid profile. The fatty acidprofile for the progeny plant having the minimum linolenic acid contentand the plant having the maximum linolenic acid content are shown inTable 6. The results show that the homozygous plant having Fad2mutations and Fad3 cosuppression had a fatty acid profile in the fieldthat was similar to that of the greenhouse-grown seed (Table 5),indicating that the Fad3 cosuppression trait and the chemically-inducedFad2 mutants conferred a stable fatty acid composition on seeds of thisplant. Thus, an oil of the invention can be obtained from eitherfield-grown seeds or greenhouse-grown seeds.

Because of the decreased α-linolenic acid content and increased oleicacid content, an oil of the invention is useful in food and industrialapplications. Oils which are low in α-linolenic acid have increasedoxidative stability. The rate of oxidation of lipid fatty acidsincreases with higher levels of linolenic acid leading to off-flavorsand off-odors in foods. The present invention provides novel canola oilsthat are low in α-linolenic acid.

To the extent not already indicated, it will be understood by those ofordinary skill in the art that any one of the various specificembodiments herein described and illustrated may be further modified toincorporate features shown in other of the specific embodiments.

The foregoing detailed description has been provided for a betterunderstanding of the invention only and no unnecessary limitation shouldbe understood therefrom as some modifications will be apparent to thoseskilled in the art without deviating from the spirit and scope of theappended claims.

6 1155 base pairs nucleic acid single linear DNA NO NO Brassica napusWild type F form. 1 ATG GGT GCA GGT GGA AGA ATG CAA GTG TCT CCT CCC TCCAAG AAG TCT 48 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser LysLys Ser 1 5 10 15 GAA ACC GAC ACC ATC AAG CGC GTA CCC TGC GAG ACA CCGCCC TTC ACT 96 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro ProPhe Thr 20 25 30 GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CAC TGT TTC AAACGC TCG 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe Lys ArgSer 35 40 45 ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATC ATC ATA GCCTCC 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile Ile Ala Ser50 55 60 TGC TTC TAC TAC NTC GCC ACC ACT TAC TTC CCT CTC CTC CCT CAC CCT240 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu Pro His Pro 6570 75 80 CTC TCC TAC TTC GCC TGG CCT CTC TAC TGG GCC TGC CAA GGG TGC GTC288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln Gly Cys Val 8590 95 CTA ACC GGC GTC TGG GTC ATA GCC CAC GAA TGC GGC CAC CAC GCC TTC336 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly His His Ala Phe 100105 110 AGC GAC TAC CAG TGG CTT GAC GAC ACC GTC GGT CTC ATC TTC CAC TCC384 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe His Ser 115120 125 TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGC AGC CAC432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg Ser His 130135 140 CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTC CCC AAG480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145150 155 160 AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AAC AAC CCTTTG 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu165 170 175 GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGC TGG CCGTTG 576 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu180 185 190 TAC TTA GCC TTC AAC GTC TCG GGA AGA CCT TAC GAC GGC GGC TTCCGT 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg195 200 205 TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGC GAG CGTCTC 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu210 215 220 CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGC TAC GGTCTC 720 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu225 230 235 240 TTC CGT TAC GCC GCC GGC CAG GGA GTG GCC TCG ATG GTC TGCTTC TAC 768 Phe Arg Tyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys PheTyr 245 250 255 GGA GTC CCG CTT CTG ATT GTC AAT GGT TTC CTC GTG TTG ATCACT TAC 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile ThrTyr 260 265 270 TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAC GAT TCG TCCGAG TGG 864 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser GluTrp 275 280 285 GAT TGG TTC AGG GGA GCT TTG GCT ACC GTT GAC AGA GAC TACGGA ATC 912 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr GlyIle 290 295 300 TTG AAC AAG GTC TTC CAC AAT ATT ACC GAC ACG CAC GTG GCCCAT CAT 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala HisHis 305 310 315 320 CCG TTC TCC ACG ATG CCG CAT TAT CAC GCG ATG GAA GCTACC AAG GCG 1008 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala ThrLys Ala 325 330 335 ATA AAG CCG ATA CTG GGA GAG TAT TAT CAG TTC GAT GGGACG CCG GTG 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp Gly ThrPro Val 340 345 350 GTT AAG GCG ATG TGG AGG GAG GCG AAG GAG TGT ATC TATGTG GAA CCG 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys Ile Tyr ValGlu Pro 355 360 365 GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGG TAC AACAAT AAG TTA T 1153 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe Trp Tyr AsnAsn Lys Leu 370 375 380 GA 1155 384 amino acids amino acid linearprotein 2 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser Lys LysSer 1 5 10 15 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr Pro ProPhe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys Phe LysArg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile Ile IleAla Ser 50 55 60 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu Leu ProHis Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys GlnGly Cys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His Glu Cys Gly HisHis Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly LeuIle Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys TyrSer His Arg Ser His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu Arg AspGlu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys Trp TyrGly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met Leu ThrVal Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe Asn ValSer Gly Arg Pro Tyr Asp Gly Gly Phe Arg 195 200 205 Cys His Phe His ProAsn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gln Ile Tyr IleSer Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 Phe ArgTyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255 GlyVal Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265 270Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275 280285 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile 290295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala His His305 310 315 320 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu Ala ThrLys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe Asp GlyThr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys IleTyr Val Glu Pro 355 360 365 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe TrpTyr Asn Asn Lys Leu 370 375 380 1155 base pairs nucleic acid singlelinear DNA NO NO Brassica napus IMC129 G to A transversion mutation atnucleotide 316 of the D form. 3 ATG GGT GCA GGT GGA AGA ATG CAA GTG TCTCCT CCC TCC AAA AAG TCT 48 Met Gly Ala Gly Gly Arg Met Gln Val Ser ProPro Ser Lys Lys Ser 1 5 10 15 GAA ACC GAC AAC ATC AAG CGC GTA CCC TGCGAG ACA CCG CCC TTC ACT 96 Glu Thr Asp Asn Ile Lys Arg Val Pro Cys GluThr Pro Pro Phe Thr 20 25 30 GTC GGA GAA CTC AAG AAA GCA ATC CCA CCG CACTGT TTC AAA CGC TCG 144 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His CysPhe Lys Arg Ser 35 40 45 ATC CCT CGC TCT TTC TCC TAC CTC ATC TGG GAC ATCATC ATA GCC TCC 192 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile IleIle Ala Ser 50 55 60 TGC TTC TAC TAC GTC GCC ACC ACT TAC TTC CCT CTC CTCCCT CAC CCT 240 Cys Phe Tyr Tyr Val Ala Thr Thr Tyr Phe Pro Leu Leu ProHis Pro 65 70 75 80 CTC TCC TAC TTC GCC TGG CCT CTC TAC TGG GCC TGC CAGGGC TGC GTC 288 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala Cys Gln GlyCys Val 85 90 95 CTA ACC GGC GTC TGG GTC ATA GCC CAC AAG TGC GGC CAC CACGCC TTC 336 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys Gly His His AlaPhe 100 105 110 AGC GAC TAC CAG TGG CTG GAC GAC ACC GTC GGC CTC ATC TTCCAC TCC 384 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val Gly Leu Ile Phe HisSer 115 120 125 TTC CTC CTC GTC CCT TAC TTC TCC TGG AAG TAC AGT CAT CGACGC CAC 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp Lys Tyr Ser His Arg ArgHis 130 135 140 CAT TCC AAC ACT GGC TCC CTC GAG AGA GAC GAA GTG TTT GTCCCC AAG 480 His Ser Asn Thr Gly Ser Leu Glu Arg Asp Glu Val Phe Val ProLys 145 150 155 160 AAG AAG TCA GAC ATC AAG TGG TAC GGC AAG TAC CTC AACAAC CCT TTG 528 Lys Lys Ser Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn AsnPro Leu 165 170 175 GGA CGC ACC GTG ATG TTA ACG GTT CAG TTC ACT CTC GGCTGG CCT TTG 576 Gly Arg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly TrpPro Leu 180 185 190 TAC TTA GCC TTC AAC GTC TCG GGG AGA CCT TAC GAC GGCGGC TTC GCT 624 Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly GlyPhe Ala 195 200 205 TGC CAT TTC CAC CCC AAC GCT CCC ATC TAC AAC GAC CGCGAG CGT CTC 672 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg GluArg Leu 210 215 220 CAG ATA TAC ATC TCC GAC GCT GGC ATC CTC GCC GTC TGCTAC GGT CTC 720 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys TyrGly Leu 225 230 235 240 TAC CGC TAC GCT GCT GTC CAA GGA GTT GCC TCG ATGGTC TGC TTC TAC 768 Tyr Arg Tyr Ala Ala Val Gln Gly Val Ala Ser Met ValCys Phe Tyr 245 250 255 GGA GTT CCG CTT CTG ATT GTC AAT GGG TTC TTA GTTTTG ATC ACT TAC 816 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val LeuIle Thr Tyr 260 265 270 TTG CAG CAC ACG CAT CCT TCC CTG CCT CAC TAT GACTCG TCT GAG TGG 864 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp SerSer Glu Trp 275 280 285 GAT TGG TTG AGG GGA GCT TTG GCC ACC GTT GAC AGAGAC TAC GGA ATC 912 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val Asp Arg AspTyr Gly Ile 290 295 300 TTG AAC AAG GTC TTC CAC AAT ATC ACG GAC ACG CACGTG GCG CAT CAC 960 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His ValAla His His 305 310 315 320 CTG TTC TCG ACC ATG CCG CAT TAT CAT GCG ATGGAA GCT ACG AAG GCG 1008 Leu Phe Ser Thr Met Pro His Tyr His Ala Met GluAla Thr Lys Ala 325 330 335 ATA AAG CCG ATA CTG GGA GAG TAT TAT CAG TTGCAT GGG ACG CCG GTG 1056 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Leu HisGly Thr Pro Val 340 345 350 GTT AAG GCG ATG TGG AGG GAG GCG AAG GAG TGTATC TAT GTG GAA CCG 1104 Val Lys Ala Met Trp Arg Glu Ala Lys Glu Cys IleTyr Val Glu Pro 355 360 365 GAC AGG CAA GGT GAG AAG AAA GGT GTG TTC TGGTAC AAC AAT AAG TTA T 1153 Asp Arg Gln Gly Glu Lys Lys Gly Val Phe TrpTyr Asn Asn Lys Leu 370 375 380 GA 1155 384 amino acids amino acidlinear protein 4 Met Gly Ala Gly Gly Arg Met Gln Val Ser Pro Pro Ser LysLys Ser 1 5 10 15 Glu Thr Asp Thr Ile Lys Arg Val Pro Cys Glu Thr ProPro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala Ile Pro Pro His Cys PheLys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr Leu Ile Trp Asp Ile IleIle Ala Ser 50 55 60 Cys Phe Tyr Tyr Xaa Ala Thr Thr Tyr Phe Pro Leu LeuPro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp Pro Leu Tyr Trp Ala CysGln Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val Ile Ala His Lys Cys GlyHis His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp Leu Asp Asp Thr Val GlyLeu Ile Phe His Ser 115 120 125 Phe Leu Leu Val Pro Tyr Phe Ser Trp LysTyr Ser His Arg Ser His 130 135 140 His Ser Asn Thr Gly Ser Leu Glu ArgAsp Glu Val Phe Val Pro Lys 145 150 155 160 Lys Lys Ser Asp Ile Lys TrpTyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 Gly Arg Thr Val Met LeuThr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190 Tyr Leu Ala Phe AsnVal Ser Gly Arg Pro Tyr Asp Gly Gly Phe Arg 195 200 205 Cys His Phe HisPro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210 215 220 Gln Ile TyrIle Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu 225 230 235 240 PheArg Tyr Ala Ala Gly Gln Gly Val Ala Ser Met Val Cys Phe Tyr 245 250 255Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val Leu Ile Thr Tyr 260 265270 Leu Gln His Thr His Pro Ser Leu Pro His Tyr Asp Ser Ser Glu Trp 275280 285 Asp Trp Phe Arg Gly Ala Leu Ala Thr Val Asp Arg Asp Tyr Gly Ile290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr Asp Thr His Val Ala HisHis 305 310 315 320 Pro Phe Ser Thr Met Pro His Tyr His Ala Met Glu AlaThr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly Glu Tyr Tyr Gln Phe AspGly Thr Pro Val 340 345 350 Val Lys Ala Met Trp Arg Glu Ala Lys Glu CysIle Tyr Val Glu Pro 355 360 365 Asp Arg Gln Gly Glu Lys Lys Gly Val PheTrp Tyr Asn Asn Lys Leu 370 375 380 1155 base pairs nucleic acid singlelinear DNA NO NO Brassica napus Wild type D form. 5 ATG GGT GCA GGT GGAAGA ATG CAA GTG TCT CCT CCC TCC AAA AAG TCT 48 Met Gly Ala Gly Gly ArgMet Gln Val Ser Pro Pro Ser Lys Lys Ser 1 5 10 15 GAA ACC GAC AAC ATCAAG CGC GTA CCC TGC GAG ACA CCG CCC TTC ACT 96 Glu Thr Asp Asn Ile LysArg Val Pro Cys Glu Thr Pro Pro Phe Thr 20 25 30 GTC GGA GAA CTC AAG AAAGCA ATC CCA CCG CAC TGT TTC AAA CGC TCG 144 Val Gly Glu Leu Lys Lys AlaIle Pro Pro His Cys Phe Lys Arg Ser 35 40 45 ATC CCT CGC TCT TTC TCC TACCTC ATC TGG GAC ATC ATC ATA GCC TCC 192 Ile Pro Arg Ser Phe Ser Tyr LeuIle Trp Asp Ile Ile Ile Ala Ser 50 55 60 TGC TTC TAC TAC GTC GCC ACC ACTTAC TTC CCT CTC CTC CCT CAC CCT 240 Cys Phe Tyr Tyr Val Ala Thr Thr TyrPhe Pro Leu Leu Pro His Pro 65 70 75 80 CTC TCC TAC TTC GCC TGG CCT CTCTAC TGG GCC TGC CAG GGC TGC GTC 288 Leu Ser Tyr Phe Ala Trp Pro Leu TyrTrp Ala Cys Gln Gly Cys Val 85 90 95 CTA ACC GGC GTC TGG GTC ATA GCC CACGAG TGC GGC CAC CAC GCC TTC 336 Leu Thr Gly Val Trp Val Ile Ala His GluCys Gly His His Ala Phe 100 105 110 AGC GAC TAC CAG TGG CTG GAC GAC ACCGTC GGC CTC ATC TTC CAC TCC 384 Ser Asp Tyr Gln Trp Leu Asp Asp Thr ValGly Leu Ile Phe His Ser 115 120 125 TTC CTC CTC GTC CCT TAC TTC TCC TGGAAG TAC AGT CAT CGA CGC CAC 432 Phe Leu Leu Val Pro Tyr Phe Ser Trp LysTyr Ser His Arg Arg His 130 135 140 CAT TCC AAC ACT GGC TCC CTC GAG AGAGAC GAA GTG TTT GTC CCC AAG 480 His Ser Asn Thr Gly Ser Leu Glu Arg AspGlu Val Phe Val Pro Lys 145 150 155 160 AAG AAG TCA GAC ATC AAG TGG TACGGC AAG TAC CTC AAC AAC CCT TTG 528 Lys Lys Ser Asp Ile Lys Trp Tyr GlyLys Tyr Leu Asn Asn Pro Leu 165 170 175 GGA CGC ACC GTG ATG TTA ACG GTTCAG TTC ACT CTC GGC TGG CCT TTG 576 Gly Arg Thr Val Met Leu Thr Val GlnPhe Thr Leu Gly Trp Pro Leu 180 185 190 TAC TTA GCC TTC AAC GTC TCG GGGAGA CCT TAC GAC GGC GGC TTC GCT 624 Tyr Leu Ala Phe Asn Val Ser Gly ArgPro Tyr Asp Gly Gly Phe Ala 195 200 205 TGC CAT TTC CAC CCC AAC GCT CCCATC TAC AAC GAC CGC GAG CGT CTC 672 Cys His Phe His Pro Asn Ala Pro IleTyr Asn Asp Arg Glu Arg Leu 210 215 220 CAG ATA TAC ATC TCC GAC GCT GGCATC CTC GCC GTC TGC TAC GGT CTC 720 Gln Ile Tyr Ile Ser Asp Ala Gly IleLeu Ala Val Cys Tyr Gly Leu 225 230 235 240 TAC CGC TAC GCT GCT GTC CAAGGA GTT GCC TCG ATG GTC TGC TTC TAC 768 Tyr Arg Tyr Ala Ala Val Gln GlyVal Ala Ser Met Val Cys Phe Tyr 245 250 255 GGA GTT CCG CTT CTG ATT GTCAAT GGG TTC TTA GTT TTG ATC ACT TAC 816 Gly Val Pro Leu Leu Ile Val AsnGly Phe Leu Val Leu Ile Thr Tyr 260 265 270 TTG CAG CAC ACG CAT CCT TCCCTG CCT CAC TAT GAC TCG TCT GAG TGG 864 Leu Gln His Thr His Pro Ser LeuPro His Tyr Asp Ser Ser Glu Trp 275 280 285 GAT TGG TTG AGG GGA GCT TTGGCC ACC GTT GAC AGA GAC TAC GGA ATC 912 Asp Trp Leu Arg Gly Ala Leu AlaThr Val Asp Arg Asp Tyr Gly Ile 290 295 300 TTG AAC AAG GTC TTC CAC AATATC ACG GAC ACG CAC GTG GCG CAT CAC 960 Leu Asn Lys Val Phe His Asn IleThr Asp Thr His Val Ala His His 305 310 315 320 CTG TTC TCG ACC ATG CCGCAT TAT CAT GCG ATG GAA GCT ACG AAG GCG 1008 Leu Phe Ser Thr Met Pro HisTyr His Ala Met Glu Ala Thr Lys Ala 325 330 335 ATA AAG CCG ATA CTG GGAGAG TAT TAT CAG TTG CAT GGG ACG CCG GTG 1056 Ile Lys Pro Ile Leu Gly GluTyr Tyr Gln Leu His Gly Thr Pro Val 340 345 350 GTT AAG GCG ATG TGG AGGGAG GCG AAG GAG TGT ATC TAT GTG GAA CCG 1104 Val Lys Ala Met Trp Arg GluAla Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 GAC AGG CAA GGT GAG AAGAAA GGT GTG TTC TGG TAC AAC AAT AAG TTA T 1153 Asp Arg Gln Gly Glu LysLys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380 GA 1155 384 aminoacids amino acid linear protein 6 Met Gly Ala Gly Gly Arg Met Gln ValSer Pro Pro Ser Lys Lys Ser 1 5 10 15 Glu Thr Asp Asn Ile Lys Arg ValPro Cys Glu Thr Pro Pro Phe Thr 20 25 30 Val Gly Glu Leu Lys Lys Ala IlePro Pro His Cys Phe Lys Arg Ser 35 40 45 Ile Pro Arg Ser Phe Ser Tyr LeuIle Trp Asp Ile Ile Ile Ala Ser 50 55 60 Cys Phe Tyr Tyr Val Ala Thr ThrTyr Phe Pro Leu Leu Pro His Pro 65 70 75 80 Leu Ser Tyr Phe Ala Trp ProLeu Tyr Trp Ala Cys Gln Gly Cys Val 85 90 95 Leu Thr Gly Val Trp Val IleAla His Glu Cys Gly His His Ala Phe 100 105 110 Ser Asp Tyr Gln Trp LeuAsp Asp Thr Val Gly Leu Ile Phe His Ser 115 120 125 Phe Leu Leu Val ProTyr Phe Ser Trp Lys Tyr Ser His Arg Arg His 130 135 140 His Ser Asn ThrGly Ser Leu Glu Arg Asp Glu Val Phe Val Pro Lys 145 150 155 160 Lys LysSer Asp Ile Lys Trp Tyr Gly Lys Tyr Leu Asn Asn Pro Leu 165 170 175 GlyArg Thr Val Met Leu Thr Val Gln Phe Thr Leu Gly Trp Pro Leu 180 185 190Tyr Leu Ala Phe Asn Val Ser Gly Arg Pro Tyr Asp Gly Gly Phe Ala 195 200205 Cys His Phe His Pro Asn Ala Pro Ile Tyr Asn Asp Arg Glu Arg Leu 210215 220 Gln Ile Tyr Ile Ser Asp Ala Gly Ile Leu Ala Val Cys Tyr Gly Leu225 230 235 240 Tyr Arg Tyr Ala Ala Val Gln Gly Val Ala Ser Met Val CysPhe Tyr 245 250 255 Gly Val Pro Leu Leu Ile Val Asn Gly Phe Leu Val LeuIle Thr Tyr 260 265 270 Leu Gln His Thr His Pro Ser Leu Pro His Tyr AspSer Ser Glu Trp 275 280 285 Asp Trp Leu Arg Gly Ala Leu Ala Thr Val AspArg Asp Tyr Gly Ile 290 295 300 Leu Asn Lys Val Phe His Asn Ile Thr AspThr His Val Ala His His 305 310 315 320 Leu Phe Ser Thr Met Pro His TyrHis Ala Met Glu Ala Thr Lys Ala 325 330 335 Ile Lys Pro Ile Leu Gly GluTyr Tyr Gln Leu His Gly Thr Pro Val 340 345 350 Val Lys Ala Met Trp ArgGlu Ala Lys Glu Cys Ile Tyr Val Glu Pro 355 360 365 Asp Arg Gln Gly GluLys Lys Gly Val Phe Trp Tyr Asn Asn Lys Leu 370 375 380

What is claimed is:
 1. A Brassica plant containing at least onerecombinant nucleic construct, said at least one construct comprising:a) a first seed-specific regulatory sequence fragment operably linked toa wild-type microsomal delta-12 fatty acid desaturase coding sequencefragment; and b) a second seed-specific regulatory sequence fragmentoperably linked to a wild-type microsomal delta-15 fatty acid desaturasecoding sequence fragment, wherein said plant produces seeds, yielding anoil having an oleic acid content of about 86% or greater and an erucicacid content of less than about 2%, said oleic acid content and erucicacid content determined after hydrolysis of said oil.
 2. The plant ofclaim 1, wherein said plant is Brassica napus, Brassica rapa, orBrassica juncea.
 3. The plant of claim 2, wherein said plant is Brassicanapus.
 4. The plant of claim 1, wherein said oleic acid content is about86% to about 89%.
 5. The plant of claim 1, wherein said oil furthercomprises an α-linolenic acid content of about 1% to about 2%.
 6. Seedsof the plant of claim
 1. 7. The plant of claim 1, wherein said plantcontains first and second recombinant nucleic acid constructs, saidfirst construct comprising said delta-12 desaturase coding sequencefragment and said second recombinant nucleic acid construct comprisingsaid delta-15 desaturase coding sequence fragment.
 8. The plant of claim1, wherein said delta-12 desaturase coding sequence fragment comprises afull-length Brassica delta-12 desaturase coding sequence.
 9. The plantof claim 1, wherein said delta-15 desaturase coding sequence fragmentcomprises a full-length Brassica delta-15 desaturase coding sequence.10. Seeds of the plant of claim
 3. 11. A Brassica plant containing atleast one recombinant nucleic acid construct, said at least oneconstruct comprising: a) a first seed-specific regulatory sequencefragment operably linked to a wild-type microsomal delta-12 fatty aciddesaturase coding sequence fragment; and b) a second seed-specificregulatory sequence fragment operably linked to a wild-type microsomaldelta-15 fatty acid desaturase coding sequence fragment, wherein saidplant produces seeds yielding an oil having an oleic acid content of 80%or greater, an α-linolenic acid content of about 2.5% or less, and anerucic acid content of less than about 2%, said oleic acid content,linolenic acid content and erucic acid content determined afterhydrolysis of said oil.
 12. The plant of claim 11, wherein said firstand second regulatory sequence fragments are linked in sense orientationto said delta-12 and delta-15 desaturase coding sequence fragments,respectively.
 13. The plant of claim 11, wherein said plant contains afirst recombinant nucleic acid construct comprising said delta-12desaturase coding sequence fragment and a second recombinant nucleicacid construct comprising said delta-15 desaturase coding sequencefragment.
 14. The plant of claim 11, wherein said delta-12 desaturasecoding sequence fragment comprises a full-length Brassica delta-12desaturase coding sequence.
 15. The plant of claim 11, wherein saiddelta-15 desaturase coding sequence fragment comprises a full-lengthBrassica delta-15 desaturase coding sequence.
 16. The plant of claim 11,wherein said plant produces seeds yielding an oil having an oleic acidcontent of about 84% to about 89%, an α-linolenic acid content of about1% to about 2%, and an erucic acid content of less than about 2%, saidoleic acid content, linolenic acid content and erucic acid contentdetermined after hydrolysis of said oil.
 17. The plant of claim 16,wherein said oleic acid content is from about 86% to about 89% and saidα-linolenic acid content is from about 1% to about 1.7%.
 18. The plantof claim 11, wherein said plant is Brassica napus, Brassica rapa, orBrassica juncea.
 19. The plant of claim 18, wherein said plant isBrassica napus.
 20. Seeds of the plant of claim
 19. 21. Seeds of theplant of claim
 11. 22. A method of producing an oil from Brassica seeds,said method comprising: a) crushing seeds of at least one transgenicBrassica plant, said Brassica plant containing at least one recombinantnucleic acid construct, said at least one construct comprising a firstseed-specific regulatory sequence fragment operably linked to awild-type delta-12 fatty acid desaturase coding sequence fragment and asecond seed-specific regulatory sequence fragment operably linked to awild-type delta-15 fatty acid desaturase coding sequence fragment; andb) extracting said oil from said crushed seeds, said oil having an oleicacid content of about 80% or greater, an α-linolenic acid content of2.5% or less, and an erucic acid content of less than 2%, said oleicacid, linolenic acid, and erucic acid content determined afterhydrolysis of said oil.
 23. The method of claim 22, said oil having anoleic acid content of about 84% to about 89%.
 24. The method of claim22, said oil having an α-linolenic acid content of about 1% to about 2%.